Nanofibrillar structure and applications including cell and tissue culture

ABSTRACT

A nanofibrillar structure for cell culture and tissue engineering is disclosed. The nanofibrillar structure can be used in a variety of applications including methods for proliferating and/or differentiating cells and manufacturing a tissue. Also disclosed is an improved nanofiber comprising a lipid, lipophilic molecule, or chemically modified surface. The nanofibers can be used in a variety of applications including the formation of nanofibrillar structures for cell culture and tissue engineering.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/703,169, filed Nov. 5, 2003. now U.S. Pat. No.7,704,740, of which the entire disclosure hereby incorporated byreference.

FIELD OF THE INVENTION

The invention relates to a nanofibrillar structure for cell culture andtissue engineering and methods for proliferating and/or differentiatingcells and manufacturing a tissue. Another aspect of the inventionrelates to a growth media for cell culture comprising a matrix ofnanofibers. Another aspect of the invention relates to an improvednanofiber comprising a lipid, lipophilic molecule, or chemicallymodified surface. The improved nanofiber is useful in a variety ofapplications. In one application, a nanofibrillar structure for cellculture and tissue engineering may be prepared using the improvednanofiber. In another application, a media for cell culture may beprepared using the improved nanofiber.

BACKGROUND OF THE INVENTION

Cell proliferation and differentiation in vivo is regulated by uniquespatial interactions between cells. Spatial cues in conjunction with thetopologically distinct location of specific attachment molecules, andthe release of specific humoral factors, such as growth anddifferentiation factors, function as signals to the cell to proliferate,differentiate, migrate, remain in a resting state, or initiateapoptosis. The capacity of the cell to respond to these signalingtriggers is dependent on the availability of specific cell surface andintracellular receptors. The signal transduction pathways that arestimulated by these molecules depend on the organization and structureof the cell cytoskeleton whose architecture is a function of multipointcell surface interactions with these signaling molecules, surroundingcells, and extracellular matrix.

In designing cell and tissue culture environments, it is important toconsider the cellular interactions that must be incorporated into thegrowth environment. Cell types, spatial cues, and chemical triggers andmodulators play a significant role in regulating gene expression withininteracting cells (Li et al., 2002, FASEB J., 17:97-99; Botarro et al.,2002, Ann. N.Y. Acad. Sci., 961:143-153; Kunz-Schughart et al., 2003,Am. J. Physiol. Cell Physiol., 284:C209-C219; Cukierman et al., 2001,Science, 294:1708-1712). Past advances in the practice of cell andtissue culture have been directed toward providing the biochemical andphysical conditions that approximate the complex in vivomicroenvironment within a tissue (Cukierman et al., 2001, Science,23:1708-1712; Li et al., 2002, FASEB J., 17:97-99; Chiu et al., 2000,Proc. Natl. Acad. Sci. USA, 97:2408-2413). These efforts have beenlimited by factors that include the use of cell lines that have beencontinuously grown on and selected for their ability to proliferate onplanar culture surfaces that lack the spatial cues and chemical triggersand modulators present in tissue in vivo.

Recent work has demonstrated that the unique micro- andnano-environments resulting from spatial organization of nanofibrils inthree dimensions, such as collagen and other fibrillar elements of theextracellular matrix, is essential for tissue-like patterns of celladherence, signal transduction, and differentiated function. Theattachment and growth of cells on solid planar culture surfaces elicitsa different pattern of cellular organization from that observed forcells in tissues in vivo (Walpita and Hay, 2002, Nature Rev. Mol. Cell.Biol., 3:137-141; Cukierman et al., 2001, Science, 23:1708-1712;Mueller-Klieser, 1997, Am. J. Physiol., C1109-C1123). When grown on atypical planar cell culture surface, fibroblasts, for example, assume ahighly spread and adhering morphology in which the actin network locatedwithin the cytoplasm is organized into arrays of thick stress fibers. Incontrast, when fibroblasts are grown within collagen gels or areobserved in tissues, they are spindle-like in shape with actin organizedin a cortical ring (Tamariz and Grinnell, 2002, Mol. Biol. Cell,13:3915-3929; Walpita and Hay, 2002, Nature Rev. Mol. Cell. Biol.,3:137-141; Grinnell et al., 2003, Mol. Biol. Cell, 14:384-395).Moreover, the drug sensitivity of cancer cells grown in two dimensionalcell cultures versus cancer cells grown in three-dimensional cellcultures has been shown to be considerably different; an outcome thathas significant bearing on the design of cancer therapies involvingchemotherapeutics (Mueller-Klieser, 1997, Am. J. Physiol,273:C1109-C1123; Padron et al., 2000, Crit. Rev. Oncol./Hematol.,36:141-157; Jacks and Weinberg, 2002, Cell, 111:923-925; Weaver et al.,2002, Cancer Cell, 2:205-216).

A significant development in cell culture and tissue culture has beenthe introduction of matrices composed of non-toxic and biocompatiblematerials designed to serve as scaffolds and three-dimensional spatialorganizers for dividing cells both in vitro and in vivo (U.S. Pat. Appl.No. 20020133229; U.S. Pat. Appl. No. 20020042128; U.S. Pat. Appl. No.20020094514; U.S. Pat. Appl. No. 20020090725). The goal of these designsis to provide a growth surface with in vivo tissue-like geometry andmicro- and nano-environments for cells to proliferate and differentiateinto functioning tissue or regenerate damaged structures. Thesestructures supporting functional cells can be utilized for a variety ofapplications, including repairing or replacing damaged tissue in thebody and promoting the growth of new tissues and organs.

The successful preparation of three-dimensional cell and tissue culturetechnology, however, has predominantly been a function of the expertisewithin individual laboratories and the availability of sophisticatedinstrumentation. There is a significant need for a culture mediummanufactured from simple or composite materials that provides the easeof use, uniformity, quality control, and flexibility associated with thestandard tissue culture plate. In addition, the culture medium materialand design may allow for the construction of layered assemblies ofdefined composition that more accurately reflect the organization ofcell layers in tissues. A media comprising multiple layers of finefibers separated by coarse fiber supports, such as the filter mediadisclosed in U.S. Pat. No. 5,672,399, does not provide an environmentfor growth of living cells.

SUMMARY OF THE INVENTION

A nanofibrillar structure can be manufactured from a nanofiber materialthat provides repeatable fiber and matrix dimensions, ease of use,uniformity, cell response, quality control, and flexibility. Thenanotopography, the topography of the nanofiber network of thenanofibrillar structure and the arrangement of the nanofibers of thenanofiber network in space is engineered to provide an in vitrobiomimetic substratum that is more tissue-compatible for the promotionof homotypic or heterotypic cell growth and/or cell differentiation insingle layer or multi-layered cell culture.

One aspect of the invention provides an improved nanofiber comprising alipid. The nanofiber has a diameter of less than about 1000 nm. Theimproved nanofiber is useful in a variety of applications, includingcell culture and tissue engineering.

A preferred mode of the invention involves a polymeric material combinedwith an additive composition that influences packing of the polymer suchthat electrospinning of the polymer results in the production of apolydisperse plurality of nanofibers having a greater number orpercentage of thin fibers as compared to a polydisperse plurality ofnanofibers electrospun from a polymer solution not containing theadditive composition. In an embodiment, the polymer solution comprisesfrom about 0.25% to about 15% w/w additive composition. In anotherembodiment, the polymer solution comprises from about 1% to about 10%w/w additive composition. In a preferred embodiment, the additivecomposition is a lipid. In another preferred embodiment, the lipid islysophosphatidylcholine, phosphatidylcholine, sphingomyelin,cholesterol, or mixtures thereof. The additive composition may alsofunction as a signaling molecule inducing recruitment and attachment ofcells to the fiber.

Thin fibers preferably have a diameter of about 5 nm to about 600 nm. Inan embodiment, thin fibers have a diameter of about 50 nm to about 400nm. In another embodiment, thin fibers have a diameter of about 300 nm.In another embodiment, thin fibers have a diameter of about 5 nm toabout 200 nm. In another embodiment, thin fibers have a diameter ofabout 5 nm to about 100 nm. In another embodiment, thin fibers have adiameter of about 5 nm to about 50 nm.

Nanofibers having a smaller diameter provide a surface that promotesmultiple point attachments between nanofibers and cells, acharacteristic of cell attachment to the extracellular matrix in vivo.Preferably at least about 25% of the polydisperse plurality ofnanofibers is thin fibers. In an embodiment, at least about 30% percentof the polydisperse plurality of lipid containing nanofibers are thinfibers. In another embodiment, at least about 40% of the polydisperseplurality of lipid containing nanofibers is thin fibers. In anotherembodiment, at least about 50% of polydisperse plurality of lipidcontaining nanofibers is thin fibers. In another embodiment, at leastabout 60% of polydisperse plurality of lipid containing nanofibers isthin fibers. In another embodiment, at least about 70% of polydisperseplurality of lipid containing nanofibers is thin fibers.

In an embodiment, the improved nanofibers are fabricated from apolyamide, polyester or other polymer suitable for in vivo, animal orhuman application. In another embodiment, the polyester may bepoly(ε-caprolactone), poly(lactate), or poly(glycolate). In anotherembodiment, the nanofibers are fabricated from a polymer solutioncomprising at least about 10% poly(ε-caprolactone) w/w in chloroform. Inanother embodiment, the nanofibers are fabricated from a polymersolution comprising at least about 15% poly(ε-caprolactone) w/w inchloroform.

Another preferred mode of the invention involves a nanofiber comprisingone or more bioactive molecules including, but not limited to peptide,polypeptides, lipids, carbohydrates, polysaccharides, amino acids,nucleotides, nucleic acids, polynucleotides, or hybrid mixtures thereof.Polypeptides include fibrous proteins, adhesion proteins, growthfactors, and differentiation factors. Some preferred growth factorsinclude VEGF, NGFs, PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF. Somepreferred differentiation factors include neurotrophins, colonystimulating factors, and transforming growth factors.

In one embodiment, the bioactive molecule is incorporated into thepolymer solution from which the nanofiber is fabricated. In anotherembodiment, functional groups may be attached to an outside surface ofthe nanofiber and the functionalized surfaces of the nanofiber reactedto bind one or more bioactive molecules. In one embodiment, functionalgroups are attached to an outside surface of the nanofiber using plasmadeposition. In another embodiment, functional groups are incorporatedinto the polymer solution from which the nanofiber is fabricated.

Another preferred mode of the invention involves a nanofiber comprisinga fluorescent marker. The fluorescent marker allows, for example,visualization of a nanofiber, identification of specific nanofiberswithin a nanofiber blend, identification of a chemical or physicalproperty of a nanofiber or the nano-environment surrounding thenanofiber, and evaluation of the degradation of and/or redistribution ofimplantable nanofibers and/or structures comprising nanofibers,including three-dimensional structures useful for engineering tissue.

The fluorescent marker may comprise an organic dye fluorophore. In anembodiment, the fluorophore is added to the polymer prior toelectrospinning of a nanofiber. In another embodiment, the fluorophoreis conjugated to a nanofiber via a functional group incorporated at thesurface of the nanofiber. In another embodiment, the fluorophore isconjugated to a bioactive molecule that is attached to a nanofiber. Thefluorescent marker may comprise colloidal inorganic semiconductornanocrystals. In an embodiment, the nanocrystals comprise a CdSe coreand ZnS cap. In another embodiment, the nanocrystals comprise quantumdots.

The fluorescent marker may act as an ion-sensing element. In anembodiment, the nanofiber may comprise a fluorescent marker wherein thefluorescence or fluorescent intensity of the marker is dependent uponion concentration. Such an ion-sensing element is useful to detectchanges in ion concentration including pH and calcium, sodium, orphosphate flux. In another embodiment, the fluorescent marker mayfunction as a reporter element to demonstrate complex formation betweenthe nanofiber surface and ligands including, but not limited to, DNA/RNAnucleotide sequences, carbohydrates, or peptides/amino acid sequences.This complex formation can be manifested by changes in fluorescenceemission wavelength and/or changes in energy transfer between anabsorber and emitter.

The present invention is also directed to a method of identifying achemical and/or physical property of a nanofiber. In an embodiment, afluorescent marker is assigned to a chemical or physical property of thenanofiber and the nanofiber is labeled with the assigned fluorescentmarker. Such chemical and physical properties include, but are notlimited to, fiber diameter, bioactive molecules, functional groups,dissolution or degradation rate of fiber, composition of polymercomprising the nanofiber, hydrophobicity or hydrophilicity of the fiber,solubility of the polymer comprising the nanofiber, toxicity of thepolymer, toxicity of bioactive molecules, or combinations thereof.Labeling nanofibers with a specific fluorescent marker, for example,allows for the identification of each type of fiber within a nanofiberblend or cellular array. A nanofiber may be labeled with more than onefluorescent marker in order to identify multiple chemical and/orphysical properties of the nanofiber.

Another aspect of the invention is a nanofibrillar structure comprisingone or more nanofibers and wherein the nanofibrillar structure isdefined by a network of one or more nanofibers. In an embodiment, thenanofiber network is deposited on a surface of a substrate. Thenanofiber may be fabricated from a variety of polymers or polymersystems. Preferably the polymer or polymer system is non-cytotoxic. Inan embodiment, the nanofibers comprise the improved nanofibers of theinvention. In another embodiment, the nanofibers are fabricated from apolyamide or polyester. In a further embodiment, the polyamide orpolyester is suitable for in vivo human application. In a furtherembodiment, the polyester may be poly(ε-caprolactone), poly(lactate) orpoly(glycolate). In a further embodiment, the polyamide may be a nylon6, a nylon 66, a nylon 610 or other biocompatible polyamides. In anembodiment, the film is an optically clear polyester film.

In an embodiment, the substrate comprises glass or plastic. In a furtherembodiment, the substrate is a surface of a culture container. Inanother embodiment, the substrate comprises a film. The film may bewater soluble or water insoluble. The film may be biodegradable and/orbiodissolvable. Preferably the film is non-cytotoxic. In a preferredembodiment, the film is polyvinyl alcohol film.

The nanofibrillar structures may be utilized singly or layered to form amulti-layered assembly of nanofibrillar structures for cell or tissueculture. In an embodiment, the nanofibrillar structure comprises aspacer. The spacer may function as a support structure. The spacerprovides sufficient openings to permit cells to penetrate and attach tothe nanofiber network. The spacer may be water soluble or waterinsoluble. The spacer may be porous or non-porous. The spacer may bebiodegradable and/or biodissolvable. Preferably the spacer isbiocompatible.

In an embodiment, the spacer comprises a first and second surfacewherein the first surface of the spacer contacts a surface of thenanofiber network deposited on a substrate and the second surface of thespacer contacts a surface of the substrate such that the nanofibernetwork and substrate are separated by the diameter or thickness of thespacer. In another embodiment, the spacer comprises a first and secondsurface wherein the first surface of the spacer contacts a surface of afirst nanofibrillar structure and the second surface of the spacercontacts a surface of a second nanofibrillar structure such that the twonanofibrillar structures are separated by the diameter or thickness ofthe spacer.

The nanofibrillar structure of the invention has many in vivo and exvivo uses including wound repair, growth of artificial skin, veins,arteries, tendons, ligaments, cartilage, heart valves, organ culture,treatment of burns, and bone grafts. In an embodiment, a diverse arrayof growth environments for a cell or tissue may be constructed byengineering specific chemical and physical properties into the nanofibernetwork, substrate, and/or spacers comprising the individualnanofibrillar structure elements and/or sequentially layering individualnanofibrillar structures. In certain embodiments, the unique nature ofthe environment can be obtained from the heterogeneous nature of thefiber diameter and composition. Physical properties and/orcharacteristics of the individual nanofiber, nanofibrillar structure,and nanofibrillar network including, but not limited to, texture,rugosity, adhesivity, porosity, solidity, elasticity, geometry,interconnectivity, surface to volume ratio, fiber diameter, fibersolubility/insolubility, hydrophilicity/hydrophobicity, and fibrildensity may be varied and/or modified to construct nano- and/ormicro-environments that promote a desired cellular activity, includingproliferation and/or differentiation. Specific nano- and/ormicro-environments may be engineered within individual nanofibrillarstructures or within a cellular array comprising two or morenanofibrillar structures.

Specific chemical properties and recognition motifs such aspolypeptides, lipids, carbohydrates, amino acids, nucleotides, nucleicacids, polynucleotides, or polysaccharides including, but not limited,to growth factors, differentiation factors, fibrous proteins, adhesiveproteins, glycoproteins, functional groups, adhesive compounds,deadhesive compounds, and targeting molecules may be engineered into thenanofibrillar network, substrate, and/or spacers of the individualnanofibrillar structures either isotropically or as gradients to promoteone or more selected cellular activities, including growth and/ordifferentiation. Some preferred growth factors include VEGF, NGFs,PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF. Some preferreddifferentiation factors include neurotrophins, colony stimulatingfactors, and transforming growth factors. Amino acids, peptides,polypeptides, and proteins may include any type of such molecules of anysize and complexity as well as combinations of such molecules including,but are not limited to, structural proteins, enzymes, and peptidehormones.

The present invention is also directed to methods of manufacturing atissue. In an embodiment, two or more nanofibrillar structures arelayered to form a multi-layered nanofibrillar assembly. Viable cells aredeposited on the fiber and the structure is cultured under conditionsthat promote growth, migration and/or differentiation of the depositedcells. In a further embodiment, nano- and/or micro-environments thatpromote cellular activity may be engineered within an individual matrixby varying and/or modifying selected physical and/or chemical propertiesof the growth matrix.

In another embodiment, multiple cell types are cultured on individualnanofibrillar structures under different culture conditions. Two or moreof the individual nanofibrillar structures are then layered to form amulti-layered nanofibrillar assembly and the assembly is cultured underconditions that promote a desired cellular activity, including growthand/or differentiation of the cells. In a further embodiment, nano-and/or micro-environments that promote cellular activity may beengineered within an individual nanofibrillar structure by varyingand/or modifying selected physical and/or chemical properties of thenanofibrillar structure or within the nanofibrillar assembly byselectively layering the individual nanofibrillar structures to obtainthe desired nano- or micro-environment. Homogeneous or heterogeneousfiber diameters and compositions may be selected to optimizeproliferation and/or differentiation.

Another aspect of the invention is a cell growth media. In anembodiment, the cell growth media comprises a matrix of nanofiberswherein the network has a fiber diameter of about 50 nm to about 1000nm, an average interfiber spacing of at least about 2 microns, a matrixsolidity of about 30 percent, and a top and a bottom with an outerwallwherein the outerwall has a height of about 10 microns to about 100 mmand wherein the top and bottom independently have an area of about 5 mm²to about 4×10⁵ mm². In another embodiment, the height of the outerwalland the area of the top and the bottom are adapted to the dimensions ofan available culture vessel or container.

The cell growth media may comprise a matrix, network, mat, sheet, orroll. In an embodiment, the cell growth media comprises a network ofnanofibers. In another embodiment, the network of nanofibers is adaptedfor insertion into a culture container. In another embodiment, the cellgrowth media is deposited onto an inside surface of a culture container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are scanning electron microscope images of nanofibrillarstructures. FIGS. 1A and B show that nanofibrillar mats composed ofrandomly deposited polyamide nanofibers can be assembled into layeredsurfaces. FIG. 1C shows that nanofibers may be electrospun with specificorientation.

FIGS. 2A-E are photomicrographs showing a comparison of microfiberselectrospun from polymer solutions comprising increasing amounts of alipid.

FIGS. 3A and B are photomicrographs showing a comparison of normal ratkidney fibroblasts grown on tissue culture plates coated with nanofiberscomprising a lipid or nanofibers not comprising a lipid.

FIGS. 4A-C are photomicrographs showing a comparison of normal ratkidney fibroblasts grown on polyamide nanofiber network, glass, andglass coated with polylysine.

FIGS. 5A and B are photomicrographs showing that incorporation of aminofunctional groups onto the surface of nanofibers.

FIG. 6 is a photomicrograph showing the fluorescent labeling ofnanofibers with quantum dots.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “nanofibrillar structure” as used herein means a structurecomprising an environment for growth of living cells comprising one ormore nanofibers, wherein the structure is defined by a network of one ormore nanofibers. In some embodiments, the nanofibrillar structurecomprises a substrate wherein the nanofibrillar structure is defined bya network of one or more nanofibers deposited on a surface of thesubstrate. The nanotopography, the topography of the nanofiber networkand the arrangement of the nanofibers of the nanofiber network in space,of the nanofibrillar structure is engineered to provide an in vitrobiomimetic substratum that is more tissue compatible for the promotionof homotypic or heterotypic cell growth and/or cell differentiation insingle layer or multi-layered cell culture. The nanofibrillar structuresmay be layered to form a multi-layered nanofibrillar assembly, cellulararray, or tissue structure.

The term “nanofiber” as used herein means a polymer fine fibercomprising a diameter of about 1000 nanometers or less. The polymer ispreferably a non-cytotoxic polymer. The polymer may be water soluble orwater insoluble. The polymer may be biodegradable and/or biodissolvable.The polymer may be a polyester or polyamide. The polyester may be analiphatic polyester including, but not limited to polylactide,poly(glycolate), poly(ε-caprolactone), and copolymers thereof. Thepolyamide may be a polycaprolactam, nylon 6, a nylon 66, nylon 6 12 orother nylon material.

The nanofiber may comprise a lipid or lipophilic molecule including, butnot limited to, lysophosphatidylcholine, phosphatidylcholine,sphingomyelin, cholesterol, and mixtures thereof. The nanofiber maycomprise one or more bioactive molecules. Preferably one of thebioactive molecules is a peptide, polypeptide, lipid, carbohydrate,polysaccharide, amino acid, nucleotide, nucleic acid, polynucleotide, orhybrid molecule thereof. The nanofiber may comprise one or more alcohol,aldehyde, amino, carboxy, sulphydryl or photoactivatable functionalgroups. Preferably the photoactivatable group is a carbene or nitrene.

The nanofiber may comprise one or more growth factors and/ordifferentiation factors. The nanofiber may release one or more growthfactors and/or differentiation factors. The rate of release isdetermined by the rate of degradation and/or dissolution of thenanofiber.

The term “network” as used herein means a random or orienteddistribution of nanofibers in space that is controlled to form aninterconnecting net with spacing between fibers selected to promotegrowth and culture stability. The network has small spaces between thefibers comprising the network forming pores or channels in the network.The pores or channels have a diameter of about 0.01 microns to about 25microns, preferably about 2 microns to about 10 microns, through athickness. A network may comprise a single layer of nanofibers, a singlelayer formed by a continuous nanofiber, multiple layers of nanofibers,multiple layers formed by a continuous nanofiber, or mat. The networkmay be unwoven or net. A network may have a thickness of about thediameter of a single nanofiber to about 2000 nm. Physical properties ofthe network including, but not limited to, texture, rugosity,adhesivity, porosity, solidity, elasticity, geometry, interconnectivity,surface to volume ratio, fiber diameter, fiber solubility/insolubility,hydrophilicity/hydrophobicity, fibril density, and fiber orientation maybe engineered to desired parameters.

The term “substrate” as used herein means any surface on which nanofiberor network of nanofibers is deposited. The substrate may be any surfacethat offers structural support for the deposited network of nanofibers.The substrate may comprise glass or plastic. Preferably the plastic isnon-cytotoxic. The substrate may be a film or culture container.

The substrate may be water soluble or water insoluble. A substrate thatis water soluble is preferably a polyvinyl alcohol film. The substratemay be porous or non-porous. Porosity of the substrate is determined bycellular penetration. A cell is able to penetrate a porous substrate butis not able to penetrate a non-porous substrate. Preferably the pores ina porous substrate have a diameter of about 2 μm to about 10 μm. Thesubstrate may be biodegradable and/or biodissolvable. Preferably thesubstrate is biocompatible.

The substrate may comprise one or more bioactive molecules. Preferablyone of the bioactive molecules is a peptide, polypeptide, lipid,carbohydrate, polysaccharide, amino acid, nucleotide, nucleic acid,polynucleotide, or hybrid molecule thereof. The substrate may compriseone or more alcohol, aldehyde, amino, carboxy, sulphydryl orphotoactivatable functional groups. Preferably the photoactivatablegroup is a carbene or nitrene. The substrate may comprise one or moregrowth factors and/or differentiation factors. The substrate may releaseone or more growth factors and/or differentiation factors. The rate ofrelease is determined by the rate of dissolution or degradation of thesubstrate.

The term “spacer” as used herein means a layer separating a nanofiber ornanofiber network from a surface of a substrate or a surface of a firstnanofibrillar structure from a surface of a second nanofibrillarstructure such that the structures are separated by the diameter orthickness of the spacer. The spacer may comprise a polymer fine fiber orfilm. Preferably the film has a thickness of about 10 microns to about50 microns. The spacer may comprise a polymer including cellulose,starch, polyamide, polyester, or polytetrafluoroethylene. The fine fibermay comprise a microfiber. A microfiber is a polymer fine fibercomprising a diameter of about 1.0 μm to about 10 μm. The microfiber maybe unwoven or net.

The spacer may be water soluble or water insoluble. The spacer may beporous or non-porous. Porosity of the spacer is determined by cellularpenetration. A cell is able to penetrate a porous spacer but is not ableto penetrate a non-porous spacer. The spacer may be biodegradable and/orbiodissolvable. Preferably the spacer is biocompatible.

The spacer may comprise one or more bioactive molecules. Preferably oneof the bioactive molecules is a peptide, polypeptide, lipid,carbohydrate, nucleotide, nucleic acid, polynucleotide, polysaccharide,amino acid, or hybrid molecule thereof. The spacer may comprise one ormore alcohol, aldehyde, amino, carboxy, sulphydryl or photoactivatablefunctional groups. Preferably the photoactivatable group is a carbene ornitrene. The spacer may comprise one or more growth factors and/ordifferentiation factors. The spacer may release one or more growthfactors and/or differentiation factors. The rate of release isdetermined by the rate of dissolution or degradation of the spacer.

The term “bioactive molecule” as used herein means a molecule that hasan effect on a cell or tissue. The term includes human or veterinarytherapeutics, nutraceuticals, vitamins, salts, electrolytes, aminoacids, peptides, polypeptides, proteins, carbohydrates, lipids,polysaccharides, nucleic acids, nucleotides, polynucleotides,glycoproteins, lipoproteins, glycolipids, glycosaminoglycans,proteoglycans, growth factors, differentiation factors, hormones,neurotransmitters, pheromones, chalones, prostaglandins,immunoglobulins, monokines and other cytokines, humectants, minerals,electrically and magnetically reactive materials, light sensitivematerials, anti-oxidants, molecules that may be metabolized as a sourceof cellular energy, antigens, and any molecules that can cause acellular or physiological response. Any combination of molecules can beused, as well as agonists or antagonists of these molecules.Glycoaminoglycans include glycoproteins, proteoglycans, and hyaluronan.Polysaccharides include cellulose, starch, alginic acid, chytosan, orhyaluronan. Cytokines include, but are not limited to, cardiotrophin,stromal cell derived factor, macrophage derived chemokine (MDC),melanoma growth stimulatory activity (MGSA), macrophage inflammatoryproteins 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4 and 5, interleukin(IL) 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, TNF-alpha, and TNF-beta. Immunoglobulins useful in thepresent invention include, but are not limited to, IgG, IgA, IgM, IgD,IgE, and mixtures thereof. Amino acids, peptides, polypeptides, andproteins may include any type of such molecules of any size andcomplexity as well as combinations of such molecules. Examples include,but are not limited to, structural proteins, enzymes, and peptidehormones.

The term bioactive molecule also includes fibrous proteins, adhesionproteins, adhesive compounds, deadhesive compounds, and targetingcompounds. Fibrous proteins include collagen and elastin.Adhesion/deadhesion compounds include fibronectin, laminin,thrombospondin and tenascin C. Adhesive proteins include actin, fibrin,fibrinogen, fibronectin, vitronectin, laminin, cadherins, selectins,intracellular adhesion molecules 1, 2, and 3, and cell-matrix adhesionreceptors including but not limited to integrins such as α₂β₁, α₆β₁,α₇β₁, α₂β₃, and α₆β₄.

The term bioactive molecule also includes leptin, leukemia inhibitoryfactor (LIF), RGD peptide, tumor necrosis factor alpha and beta,endostatin, angiostatin, thrombospondin, osteogenic protein-1, bonemorphogenic proteins 2 and 7, osteonectin, somatomedin-like peptide,osteocalcin, interferon alpha, interferon alpha A, interferon beta,interferon gamma, interferon 1 alpha, and interleukins 2, 3, 4, 5 6, 7,8, 9, 10, 11, 12, 13, 15, 16, 17 and 18.

The term “growth factor” as used herein means a bioactive molecule thatpromotes the proliferation of a cell or tissue. Growth factors useful inthe present invention include, but are not limited to, transforminggrowth factor-alpha. (TGF-alpha), transforming growth factor-beta.(TGF-beta), platelet-derived growth factors including the AA, AB and BBisoforms (PDGF), fibroblast growth factors (FGF), including FGF acidicisoforms 1 and 2, FGF basic form 2, and FGF 4, 8, 9 and 10, nerve growthfactors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF andneurotrophins, brain derived neurotrophic factor, cartilage derivedfactor, bone growth factors (BGF), basic fibroblast growth factor,insulin-like growth factor (IGF), vascular endothelial growth factor(VEGF), EG-VEGF, VEGF-related protein, Bv8, VEGF-E, granulocyte colonystimulating factor (G-CSF), insulin like growth factor (IGF) I and II,hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stemcell factor (SCF), keratinocyte growth factor (KGF), transforming growthfactors (TGF), including TGFs alpha, beta, beta1, beta2, and beta3,skeletal growth factor, bone matrix derived growth factors, and bonederived growth factors and mixtures thereof. Some growth factors mayalso promote differentiation of a cell or tissue. TGF, for example, maypromote growth and/or differentiation of a cell or tissue. Somepreferred growth factors include VEGF, NGFs, PDGF-AA, PDGF-BB, PDGF-AB,FGFb, FGFa, and BGF.

The term “differentiation factor” as used herein means a bioactivemolecule that promotes the differentiation of cells. The term includes,but is not limited to, neurotrophin, colony stimulating factor (CSF), ortransforming growth factor. CSF includes granulocyte-CSF,macrophage-CSF, granulocyte-macrophage-CSF, erythropoietin, and IL-3.Some differentiation factors may also promote the growth of a cell ortissue. TGF and IL-3, for example, may promote differentiation and/orgrowth of cells.

The term “adhesive compound” as used herein means a bioactive moleculethat promotes attachment of a cell to a fiber surface comprising theadhesive compound. Examples of adhesive compounds include, but are notlimited to, fibronectin, vitronectin, and laminin.

The term “deadhesive compound” as used herein means a bioactive moleculethat promotes the detachment of a cell from a fiber comprising thedeadhesive compound. Examples of deadhesive compounds include, but arenot limited to, thrombospondin and tenascin C.

The term “targeting compound” as used herein means a bioactive moleculethat functions as a signaling molecule inducing recruitment and/orattachment of cells to a fiber comprising the targeting compound.Examples of targeting compounds and their cognate receptors includeattachment peptides including RGD peptide derived from fibronectin andintegrins, growth factors including EGF and EGF receptor, and hormonesincluding insulin and insulin receptor.

The term “lipid” as used herein means an organic molecule that isinsoluble in water but tends to dissolve in nonpolar organic solvents.The term includes lipophilic molecules, including, but not limited toplant and animal triglycerides, sterols, phosphatidylcholine materials,including lysophosphatidylcholine, phosphatidylcholine, sphingomyelin,and cholesterol.

The phrase “adapted for insertion” as used herein means manufactured orfabricated for use in or to the dimensions of a culture container orresizing for use in or to the dimensions of a culture containerincluding, for example, cutting down to size or cutting a piece from asheet, roll, or mat to a size suitable for insertion into a culturecontainer.

The term “culture container” as used herein means a receptacle forholding media for culturing a cell or tissue. The culture container maybe glass or plastic. Preferably the plastic is non-cytotoxic. The termculture container includes, but is not limited to, single and multiwellculture plates, chambered and multi-chambered culture slides,coverslips, cups, flasks, tubes, bottles, roller bottles, spinnerbottles, perfusion chambers, bioreactors and fermenters.

The term “mat” as used herein means a densely interwoven, tangled oradhered mass of nanofibers. The distribution of nanofibers in the matmay be random or oriented. A mat may be unwoven or net. A mat may or maynot be deposited on a substrate. A mat has a thickness of about 100 toabout 1000 nm.

II. Modes for Carrying Out the Invention A. Improved Nanofiber

One aspect of the invention provides an improved nanofiber comprising alipid. The nanofiber preferably has a diameter of less than about 1000nm. In an embodiment, the nanofiber has a diameter of about 50 to about1000 nanometers The improved nanofiber is useful in a variety ofapplications, including cell culture and tissue engineering.

i. Polymer and Polymer Systems

The improved nanofiber preferably comprises a non-cytotoxic polymer. Thepolymer may be water soluble or water insoluble. The polymer may bebiodegradable and/or biodissolvable. The polymer may comprise a firstpolymer and a second, but different polymer (differing in polymer type,molecular weight or physical property) that is conditioned or treated atelevated temperature.

The polymer blend can be reacted and formed into a single chemicalspecie or can be physically combined into a blended composition by anannealing process. Annealing implies a physical change, likecrystallinity, stress relaxation or orientation. Preferred materials arechemically reacted into a single polymeric specie such that aDifferential Scanning Calorimeter analysis reveals a single polymericmaterial. Such a material, when combined with a preferred additivematerial, can form a surface coating of the additive on the nanofiberthat provides oleophobicity, hydrophobicity or other associated improvedstability when contacted with high temperature, high humidity anddifficult operating conditions. The fine fiber of the class of materialscan have a diameter of about 1000 nm to less than about 5 nanometers.Such fibers can have a smooth surface comprising a discrete layer of theadditive material or an outer coating of the additive material that ispartly solubilized or alloyed in the polymer surface, or both. Preferredmaterials for use in the blended polymeric systems include nylon 6;nylon 66; nylon 6-10; nylon (6-66-610) copolymers and other lineargenerally aliphatic nylon compositions. A preferred nylon copolymerresin (SVP-651) was analyzed for molecular weight by the end grouptitration. (J. E. Walz and G. B. Taylor, determination of the molecularweight of nylon, Anal. Chem. Vol. 19, Number 7, pp 448-450 (1947). Anumber average molecular weight (W_(n)) was between 21,500 and 24,800.The composition was estimated by the phase diagram of melt temperatureof three component nylon, nylon 6 about 45%, nylon 66 about 20% andnylon 610 about 25%. (Page 286, Nylon Plastics Handbook, Melvin Kohaned. Hanser Publisher, New York (1995)).

Reported physical properties of SVP 651 resin are:

Property ASTM Method Units Typical Value Specific Gravity D-792 —  1.08Water Absorption D-570 %  2.5 (24 hr immersion) Hardness D-240 Shore D 65 Melting Point DSC ° C. (° F.) 154 (309) Tensile Strength D-638 MPa(kpsi)  50 (7.3) @ Yield Elongation at Break D-638 % 350 FlexuralModulus D-790 MPa (kpsi) 180 (26) Volume Resistivity D-257 ohm-cm  10¹²

A polyvinyl alcohol having a hydrolysis degree of from 87 to 99.9+% canbe used in such polymer systems. These are preferably crosslinked, andthey are most preferably crosslinked and combined with substantialquantities of the oleophobic and hydrophobic additive materials.

The polymer may be a single polymeric material optionally combined withan additive composition to improve fiber lifetime or operationalproperties. The preferred polymers useful in this aspect of theinvention include nylon polymers, polyvinylidene chloride polymers,polyvinylidene fluoride polymers, polyvinyl alcohol polymers and, inparticular, those listed materials when combined with stronglyoleophobic and hydrophobic additives that can result in a microfiber ornanofiber with the additive materials formed in a coating on the finefiber surface. Again, blends of similar polymers such as a blend ofsimilar nylons, similar polyvinylchloride polymers, blends ofpolyvinylidene chloride polymers are useful in this invention. Further,polymeric blends or alloys of differing polymers are also contemplatedby the invention. In this regard, compatible mixtures of polymers areuseful in forming the nanofiber or microfiber materials of theinvention.

Additive compositions may be organic or inorganic, metals or non-metals.In an embodiment, the polymer solution comprises from about 0.25 percentto about 70 percent w/w additive composition. In a further embodiment,the additive composition is a bioactive molecule. In another furtherembodiment, the additive composition is a ceramic. The additivecomposition may be an optical additive that increases or decreasesinherent fiber fluorescence for microscopy. In an embodiment, theoptical additive is a quantum dot. In another embodiment, the opticaladditive minimizes fluorescent background of the fiber by enhancing thesignal to noise ratio. Examples of optical additives include, but arenot limited to quantum dots or Fluoroblok™ (Bectin Dickinson, FranklinLakes, N.J.).

Polymer materials that can be used in the polymeric compositions of theinvention include both addition polymer and condensation polymermaterials such as polyolefin, polyacetal, polyamide, polyester,cellulose ether and ester, polyalkylene sulfide, polyarylene oxide,polysulfone, modified polysulfone polymers and mixtures thereof.Preferred materials that fall within these generic classes includepolyethylene, poly(ε-caprolactone), poly(lactate), poly(glycolate),polypropylene, poly(vinylchloride), polymethylmethacrylate (and otheracrylic resins), polystyrene, and copolymers thereof (including ABA typeblock copolymers), poly(vinylidene fluoride), poly(vinylidene chloride),polyvinyl alcohol in various degrees of hydrolysis (87% to 99.5%) incrosslinked and non-crosslinked forms. Preferred addition polymers tendto be glassy (a Tg greater than room temperature). This is the case forpolyvinylchloride and polymethylmethacrylate, polystyrene polymercompositions or alloys or low in crystallinity for polyvinylidenefluoride and polyvinyl alcohol materials.

Aliphatic polyesters such as poly(ε-caprolactone), poly(lactate),poly(glycolate), and their copolymers are biodegradable, andbiocompatible and among the few synthetic polymers approved by the Foodand Drug Administration (FDA) for certain human clinical applicationssuch as surgical sutures and some implantable devices. In an embodiment,the nanofibers are fabricated from an aliphatic polyester suitable forin vivo human application. Preferably the polyester ispoly(ε-caprolactone), poly(lactate) or poly(glycolate). In anembodiment, the nanofibers are fabricated from a polymer solutioncomprising at least about 10% poly(ε-caprolactone) w/w in chloroform. Inanother embodiment, the nanofibers are fabricated from a polymersolution comprising at least about 15% poly(ε-caprolactone) w/w inchloroform.

One class of polyamide condensation polymers are nylon materials. Theterm “nylon” is a generic name for all long chain synthetic polyamides.Typically, nylon nomenclature includes a series of numbers such as innylon-6,6 which indicates that the starting materials are a C₆ diamineand a C₆ diacid (the first digit indicating a C₆ diamine and the seconddigit indicating a C₆ dicarboxylic acid compound). Another nylon can bemade by the polycondensation of epsilon caprolactam in the presence of asmall amount of water. This reaction forms a nylon-6 (made from a cycliclactam—also known as epsilon-aminocaproic acid) that is a linearpolyamide. Further, nylon copolymers are also contemplated. Copolymerscan be made by combining various diamine compounds, various diacidcompounds and various cyclic lactam structures in a reaction mixture andthen forming the nylon with randomly positioned monomeric materials in apolyamide structure. For example, a nylon 6,6-6,10 material is a nylonmanufactured from hexamethylene diamine and a C₆ and a C₁₀ blend ofdiacids. A nylon 6-6,6-6,10 is a nylon manufactured by copolymerizationof epsilon aminocaproic acid, hexamethylene diamine and a blend of a C₆and a C₁₀ diacid material.

Block copolymers are also useful in the process of this invention. Withsuch copolymers the choice of solvent swelling agent is important. Theselected solvent is such that both blocks were soluble in the solvent.One example is a ABA (styrene-EP-styrene) or AB (styrene-EP) polymer inmethylene chloride solvent. If one component is not soluble in thesolvent, it will form a gel. Examples of such block copolymers areKraton® type of AB and ABA block polymers including styrene/butadieneand styrene/hydrogenated butadiene(ethylene propylene), Pebax® type ofepsilon-caprolactam/ethylene oxide, Sympatex® polyester/ethylene oxideand polyurethanes of ethylene oxide and isocyanates.

Addition polymers like polyvinylidene fluoride, syndiotacticpolystyrene, copolymer of vinylidene fluoride and hexafluoropropylene,polyvinyl alcohol, polyvinyl acetate, amorphous addition polymers, suchas poly(acrylonitrile) and its copolymers with acrylic acid andmethacrylates, polystyrene, poly(vinyl chloride) and its variouscopolymers, poly(methyl methacrylate) and its various copolymers, can besolution spun with relative ease because they are soluble at lowpressures and temperatures. However, highly crystalline polymer likepolyethylene and polypropylene require high temperature, high pressuresolvent if they are to be solution spun. Therefore, solution spinning ofthe polyethylene and polypropylene is very difficult. Electrostaticsolution spinning is one method of making nanofibers and microfiber.

We have also found a substantial advantage to forming polymericcompositions comprising two or more polymeric materials in polymeradmixture, alloy format or in a crosslinked chemically bonded structure.We believe such polymer compositions improve physical properties bychanging polymer attributes such as improving polymer chain flexibilityor chain mobility, increasing overall molecular weight and providingreinforcement through the formation of networks of polymeric materials.

In one embodiment of this concept, two related polymer materials can beblended for beneficial properties. For example, a high molecular weightpolyvinylchloride can be blended with a low molecular weightpolyvinylchloride. Similarly, a high molecular weight nylon material canbe blended with a low molecular weight nylon material. Further,differing species of a general polymeric genus can be blended. Forexample, a high molecular weight styrene material can be blended with alow molecular weight, high impact polystyrene. A Nylon-6 material can beblended with a nylon copolymer such as a Nylon-6; 6,6; 6,10 copolymer.Further, a polyvinyl alcohol having a low degree of hydrolysis such as a87% hydrolyzed polyvinyl alcohol can be blended with a fully or superhydrolyzed polyvinyl alcohol having a degree of hydrolysis between 98and 99.9% and higher. All of these materials in admixture can becrosslinked using appropriate crosslinking mechanisms. Nylons can becrosslinked using crosslinking agents that are reactive with thenitrogen atom in the amide linkage. Polyvinyl alcohol materials can becrosslinked using hydroxyl reactive materials such as monoaldehydes,such as formaldehyde, ureas, melamine-formaldehyde resin and itsanalogues, boric acids and other inorganic compounds. dialdehydes,diacids, urethanes, epoxies and other known crosslinking agents.Crosslinking technology is a well known and understood phenomenon inwhich a crosslinking reagent reacts and forms covalent bonds betweenpolymer chains to substantially improve molecular weight, chemicalresistance, overall strength and resistance to mechanical degradation.

Electrospinning produces a population of nanofibers that may differ indiameter, typically from about 5 nm to about 1000 nm. A preferred modeof the invention involves a polymeric material combined with an additivecomposition that influences packing of the polymer such thatelectrospinning of the polymer results in the production of a populationof nanofibers having a greater number or percentage of thin fibers ascompared to a population of nanofibers electrospun form a polymersolution not containing the additive composition. In an embodiment, thepolymer solution comprises from about 0.25% to about 15% w/w additivecomposition. In another embodiment, the polymer solution comprises fromabout 1% to about 10% w/w additive composition.

Thin fibers preferably have a diameter of about 5 nm to about 600 nm. Inan embodiment, thin fibers have a diameter of about 50 nm to about 400nm. In another embodiment, thin fibers have a diameter of about 300 nm.In another embodiment, thin fibers have a diameter of about 5 nm toabout 200 nm. In another embodiment, thin fibers have a diameter ofabout 5 nm to about 100 nm. In another embodiment, thin fibers have adiameter of about 5 nm to about 50 nm.

Nanofibers having a smaller diameter provide a surface that promotesmultipoint attachments between nanofibers and cells, a characteristic ofcell attachment to the extracellular matrix in vivo. Preferably at leastabout 25% of the population of lipid containing nanofibers is thinfibers. In an embodiment, at least about 30% percent of the populationof lipid containing nanofibers are thin fibers. In another embodiment,at least about 40% of the polydisperse plurality of lipid containingnanofibers is thin fibers. In another embodiment, at least about 50% ofpolydisperse plurality of lipid containing nanofibers is thin fibers. Inanother embodiment, at least about 60% of polydisperse plurality oflipid containing nanofibers is thin fibers. In another embodiment, atleast about 70% of polydisperse plurality of lipid containing nanofibersis thin fibers.

Preferably the additive composition is non-cytotoxic. In an embodiment,the additive composition that influences packing of the polymer is abioactive molecule. The bioactive molecule may be a lipid. Preferablythe lipid is lysophosphatidylcholine, phosphatidylcholine,sphingomyelin, cholesterol, and mixtures thereof.

The additive composition may or may not affect the activity of cells,including migration or cell attachment to the nanofibers. In anembodiment, a nanofiber comprising the additive composition does notaffect the activity of cells. Preferably, the additive compositioncomprises one or more bioactive molecules. One or more of the bioactivemolecules may be a lipid. Preferably the lipid is cholesterol. Inanother embodiment, a nanofiber comprising the additive composition mayaffect the activity of cells. Such a nanofiber may induce cell migrationor enhance attachment of cells to the nanofiber. Preferably the additivecomposition comprises one or more bioactive molecules. One or morebioactive molecules may be a lipid. In an embodiment, the lipid islysophosphatidylcholine, phosphatidylcholine, sphingomyelin, or mixturesthereof. In another embodiment, the additive composition comprisescholesterol and one or more bioactive molecules that affect the activityof cells including growth factors, differentiation factors, and/oradhesive proteins.

The polymer or polymer system may comprise one or more bioactivemolecules including but not limited to lipids or lipophilic molecules,fibrous proteins, adhesion proteins, growth factors, and differentiationfactors. Preferably at least one of the bioactive molecules comprises alipid. In an embodiment, the lipid molecules may function as signalingmolecules inducing recruitment and attachment of cells to the fiber. Thelipid molecules may also cause the cells to proliferate ordifferentiate. Nanofibers comprising bioactive molecules, such aslipids, that promote guided migration and tight attachment of cellswhich may be used in vivo or ex vivo for applications including woundrepair, growth of artificial skin, veins, arteries, tendons, ligaments,cartilage, heart valves organ cultures, treatment of burns, and bonegrafts. Preferably the lipid is lysophosphatidylcholine,phosphatidylcholine, sphingomyelin, or mixtures thereof.

Preferably one or more of the bioactive molecules is a growth factor,differentiation factor, fibrous protein, and/or adhesive protein.Preferably the growth factor is VEGF, bone morphogenic factor β, EGF,PDGF, NGF, FGF, IGF, or TGF. Preferably the differentiation factor isneurotrophin, CSF, or TGF. Preferably the differentiation factor isneurotrophin, CSF, or TGF.

The polymer systems of the invention have adhering characteristic suchthat when contacted with a cellulosic, polyvinyl, polyester,polystyrene, or polyamide substrate adheres to the substrate withsufficient strength such that it is securely bonded to the substrate andcan resist delaminating effects associated with mechanical stresses. Thenanofibers of the invention may be used to construct three-dimensionalfunctional tissues, including muscle and tendon. In such a mode, thepolymer material must stay attached to the substrate while undergoingmechanical stresses associated with, for example, contraction of amuscle or tendon. Adhesion of the nanofiber to the substrate can arisefrom solvent effects of fiber formation as the fiber is contacted withthe substrate or the post treatment of the fiber on the substrate withheat or pressure. However, polymer characteristics appear to play animportant role in determining adhesion, such as specific chemicalinteractions like hydrogen bonding, contact between polymer andsubstrate occurring above or below Tg, and the polymer formulationincluding additives. Polymers plasticized with solvent or steam at thetime of adhesion can have increased adhesion.

ii. Functionalized Surfaces

Functional groups may be incorporated at the outside surface of thenanofibers. These functionalized surfaces may be reacted to bind apeptide, polypeptide, lipid, carbohydrate, polysaccharide, amino acid,nucleotide, nucleic acid, polynucleotide, or other bioactive molecule tothe surface of the nanofiber. In an embodiment, the functionalizedsurfaces of the nanofiber are reacted to bind one or more bioactivemolecules. Preferably one or more of the bioactive molecules is a growthfactor, differentiation factor, adhesive protein, or bioactive peptidederived from an adhesive protein. Preferably the growth factor is VEGF,bone morphogenic factor β, EGF, PDGF, NGF, FGF, IGF, or TGF. Preferablythe differentiation factor is neurotrophin, CSF, or TGF. Preferably thedifferentiation factor is neurotrophin, CSF, or TGF. Preferably thebioactive peptide is RGD peptide.

In an embodiment, functional groups are deposited on the outside surfaceof a nanofiber by plasma deposition. Plasma deposition creates localplasmas at the surface of the nanofiber. The treated surface is thenreacted with gaseous molecules, such as allylamine and/or allyl alcohol,in a reaction chamber. In another embodiment, functional groups areintroduced onto the surface of the nanofibers during the electrospinningprocess. Dodecyl amine, dodecyl aldehyde, dodecyl thiol, or dodecylalcohol may be added to the polymer solution. The polymer solution isthan electrospun into nanofibers in which a portion of the added amines,aldehydes, sulphydryl, or alcohol moieties, respectively, are exposed onthe outside surface of the nanofibers.

iii. Fluorescent Marker

The nanofibers may comprise a fluorescent marker. The fluorescent markerallows, for example, visualization of a nanofiber, identification ofspecific nanofibers within a nanofiber blend, identification of achemical or physical property of a nanofiber, and evaluation of thedegradation of and/or redistribution of implantable nanofibers and/orstructures comprising nanofibers, including multi-layered assembliesuseful for engineering tissue, which can be degraded and transported toother regions distant from the original site of implantation. Thefluorescent marker may be photobleachable or non-photobleachable. Thefluorescent marker may be pH sensitive or pH insensitive. Preferably thefluorescent marker is non-cytotoxic.

The fluorescent marker may comprise an organic dye fluorophoreincluding, but not limited to, Texas Red®, BIODIPY®, Oregon Green®,Alexa Fluor®, fluorescein, Cascade Blue®, Dapoxyl®, coumarin, Rhodamine,N-methyl-4-hydrazine-7-nitrobenzofurazan, dansyl ethylenediamine, dansylcadaverine, dansyl hydrazine, or mixtures thereof. More information onthese and other suitable organic dye fluorophores may be found atwww-probes-com (Molecular Probes, Eugene, Oreg.). In an embodiment, thefluorophore is added to the polymer prior to electrospinning of ananofiber. In another embodiment, the fluorophore is conjugated to ananofiber via a functional group incorporated at the surface of thenanofiber. In another embodiment, the fluorophore is conjugated to abioactive molecule that is attached to a nanofiber.

The fluorescent marker may comprise colloidal inorganic semiconductornanocrystals. In an embodiment the nanocrystals comprise a CdSe core andZnS cap. In another embodiment the nanocrystals comprise quantum dots.More information on nanocrystals and quantum dots may be found atwww-evidenttech-com and www-quantumdots-com. The absorption spectra ofthe nanocrystal may be broad, extending from ultraviolet to a cutoff inthe visible spectrum. The emission spectra may be narrow, preferably20-40 nm full width at half maximum centered at a wavelength that ischaracteristic of the particle size of the selected nanocrystal.Preferably the nanocrystals are photochemically stable and/ornon-cytotoxic.

The fluorescent marker may be used to identify a chemical and/orphysical property of the nanofiber. In an embodiment, a fluorescentmarker is assigned to a chemical or physical property of the nanofiberand the nanofiber is labeled with the assigned fluorescent marker. Suchchemical and physical properties include, but are not limited to, fiberdiameter, bioactive molecules, functional groups, dissolution ordegradation rate of fiber, composition of polymer comprising thenanofiber, hydrophobicity or hydrophilicity of the fiber; solubility ofthe polymer comprising the nanofiber, toxicity of the polymer, toxicityof bioactive molecules, or combinations thereof. In an embodiment, thebioactive molecule is a growth factor, differentiation factor, anadhesion molecule, or mixtures thereof. Labeling nanofibers with aspecific fluorescent marker, for example, allows for the identificationof each type of fiber within a nanofiber blend or cellular array. Ananofiber may be labeled with more than one fluorescent marker in orderto identify multiple chemical and/or physical properties of thenanofiber.

The fluorescent marker may comprise bioactive fluorescent probes todetermine changes in a biochemical environment. In an embodiment, thenanofiber may comprise a fluorescent marker wherein the fluorescence orfluorescent intensity of the marker is dependent upon ion concentration.Such an ion sensing element is useful to detect changes in ionconcentration including pH and calcium, sodium, or phosphate flux. In anembodiment, the fluorescent marker comprises SNARF, SNAFL, calciumgreen, or mixtures thereof. In another embodiment, the nanofibercontains dyes capable of changing their fluorescent properties as aresult of complexion with other molecules.

iv. Applications

The improved nanofiber may be used in many known applications employingnanofibers including, but not limited to, filter applications, computerhard drive applications, and pharmaceutical applications. The improvednanofiber is useful in a variety of biological applications, includingcell culture, tissue culture, and tissue engineering applications. Inone application, a nanofibrillar structure for cell culture and tissueengineering may be fabricated using the improved nanofiber. In anembodiment, the nanofibrillar structure comprises one or more improvednanofibers, wherein the nanofibrillar structure is defined by a networkof one or more improved nanofibers. In another embodiment, thenanofibrillar structure comprises one or more improved nanofibers and asubstrate wherein the nanofibrillar structure is defined by a network ofone or more improved nanofibers deposited on a surface of the substrate.

In another application, a growth media for cell culture may be preparedusing the improved nanofiber. In an embodiment, the growth mediacomprises a matrix of nanofibers in the form of a mat, roll, or sheetthat may be adapted for insertion into a culture container. In anotherembodiment, the growth media comprises a matrix of nanofibers that isdeposited onto a surface of a culture container or added as a fibrousmesh to the culture container.

In another application, the improved nanofiber may be sprayed or spunonto a three-dimensional structure suitable for cell or tissue culture.The resultant three-dimensional structure is returned to a cell cultureapparatus for continued growth where the electrospun fiber structureserves as a platform for growth of the cells. In a further application,the improved nanofibers may be electrospun into nonwoven mesh and/orbraids for the layered construction of three-dimensional matrices toserve as templates for tissue regeneration. In a further application,the improved nanofibers may be used as a cell culture medium in highthroughput drug analysis and drug sensitivity analysis to increase thenumber of cells per well providing higher signal for detection of cellresponse. In another further application, the improved nanofibers may beused as a cell culture medium in high throughput drug analysis, drugsensitivity analysis, and other therapeutic schemes where the nanofibersprovide an environment for the cells to more closely mimic the in vivonature of the cells in an ex vivo environment.

B. Nanofibrillar Structure

Another aspect of the invention is a nanofibrillar structure. Thenanofibrillar structure comprises an environment for growth of livingcells comprising one or more nanofibers, wherein the nanofibrillarstructure is defined by a network of one or more nanofibers. In someembodiments, the nanofibrillar structure comprises a substrate whereinthe nanofibrillar structure is defined by a network of one or morenanofibers deposited on a surface of the substrate. The nanotopographyof the nanofibrillar structure may be engineered to provide a moretissue-like substratum for the promotion of homotypic or heterotypiccell growth and/or cell differentiation in single layer or multi-layeredcell culture.

i. Nanofiber Network

The nanofibers comprising the nanofibrillar structure may comprise apolymer or polymer system as described above for the improved nanofiber.In an embodiment, the nanofibers are fabricated from a polymer suitablefor in vivo human application. The nanofiber may be fabricated by manytechniques, including preferred electrospinning techniques. Polymerselection and/or the process by which the nanofibers are fabricatedand/or directed and oriented onto a substrate allow for specificselection and manipulation of physical properties of the nanofibernetwork. Physical properties of a growth surface, including fiber size,fiber diameter, fiber spacing, matrix density, fiber texture andelasticity, have been demonstrated to be important considerations fororganizing the cytoskeletal networks in cells and the exposure of cellsignaling motifs in extracellular matrix proteins (Meiners, S, andMercado, M. L., 2003, Mol. Neurobiol., 27(2), 177-196). Physicalproperties of the nanofiber network that may be engineered to desiredparameters include, but are not limited to, texture, rugosity,adhesivity, porosity, solidity, elasticity, geometry, interconnectivity,surface to volume ratio, fiber size, fiber diameter, fibersolubility/insolubility, hydrophilicity/hydrophobicity, and fibrildensity.

One or more of the physical properties of the nanofibrillar structuremay be varied and/or modified to create a specifically definedenvironment for cell growth and/or differentiation. For example,porosity of the nanofibrillar structure may be engineered to enhancediffusion of ions, metabolites, and/or bioactive molecules and/or allowcells to penetrate and permeate the nanofibrillar structure to grow inan environment that promotes multipoint attachments between the cellsand the nanofiber network. Interconnectivity of the nanofiber network ofthe nanofibrillar structure may be engineered to facilitate cell-cellcontacts. Elasticity of the nanofiber network of the nanofibrillarstructure may be increased or decreased by adding a bioactive moleculeto the polymer solution from which the nanofibers are fabricated. In anembodiment, the bioactive molecule is a lipid. In a further embodimentthe lipid is cholesterol.

Texture and rugosity of the nanofibrillar structure may be engineered topromote attachment of cells. Homogeneous or heterogeneous nanofibercompositions may be selected to optimize growth or differentiationactivity of the cells. For example, the nanofibrillar structure may becomprised of multiple nanofibers having different diameters and/ormultiple nanofibers fabricated from different polymers. Solubility orinsolubility of the nanofibers of the nanofiber network may beengineered to control the release of bioactive molecules fromnanofibrillar structure. In an embodiment, the rate of release ofbioactive molecules is determined by the rate of biodegradation orbiodissolution of the nanofibers of the nanofiber network.Hydrophobicity and hydrophilicity of the nanofiber network of thenanofibrillar structure may be engineered to promote specific cellspacing. Solidity of the nanofibrillar structure may be engineered topromote cell growth and/or differentiation. In an embodiment, thenanofibrillar structure has a solidity of about 3 percent to about 70percent. In another embodiment, the nanofibrillar structure has asolidity of about 3 percent to about 50 percent. In another embodiment,the nanofibrillar structure has a solidity of about 3 percent to about30 percent. In another embodiment, the nanofibrillar structure has asolidity of about 3 percent to about 10 percent. In another embodiment,the nanofibrillar structure has a solidity of about 3 percent to about 5percent. In another embodiment, the nanofibrillar structure has asolidity of about 10 percent to about 30 percent.

The electrospinning process uses an electric field to control theformation and deposition of polymers. A polymer solution is injectedwith an electrical potential. The electrical potential creates a chargeimbalance that leads to the ejection of a polymer solution stream fromthe tip of an emitter such as a needle. The polymer jet within theelectric field is directed toward a grounded substrate, during whichtime the solvent evaporates and fibers are formed. The resulting singlecontinuous filament collects as a nonwoven fabric on the substrate.

Electrospun nanofiber networks may be produced having random or directedorientations. As shown in FIGS. 1A and B, random fibers may be assembledinto layered surfaces. In an embodiment, the nanofibers of the inventioncomprise a random distribution of fine fibers that can be bonded to forman interlocking network. The nanofiber interlocking networks haverelatively small spaces between the fibers. Such spaces typically range,between fibers, of about 0.01 to about 25 microns, preferably about 2 toabout 10 microns. Such spaces form pores or channels in the nanofibernetwork allowing for diffusion of ions, metabolites, proteins, and/orbioactive molecules and/or allowing cells to penetrate and permeate thenetwork and grow in an environment that promotes multipoint attachmentsbetween cells and the nanofibers.

As shown in FIG. 1C, nanofiber networks may be electrospun in anoriented manner. Such oriented electrospinning allows for thefabrication of a nanofiber network comprising a single layer ofnanofibers or a single layer formed by a continuous nanofiber whereinthe network has a height of the diameter of a single nanofiber. Physicalproperties including porosity, solidity, fibril density, texture,rugosity, and fiber orientation of the single layer network may beselected by controlling the direction and/or orientation of thenanofiber onto the substrate during the electrospinning process.Preferably the pore size allows cells to penetrate and/or migratethrough the single layer nanofiber network. In an embodiment, the spacebetween fibers is about 0.01 to about 25 microns. In another embodiment,the space between fibers is about 2 to about 10 microns.

Layering of individual single layer networks form channels in thenanofibrillar structure allowing diffusion of ions, metabolites,proteins, and/or bioactive molecules and allowing cells to penetrate thenanofibrillar structure and grow in an environment that promotesmultipoint attachments between the cells and the nanofiber network.

Phase separation techniques may also be used to fabricate thenanofibrillar structure. The phase separation process typically includespolymer dissolution, phase separation and gelatin, solvent extractionfrom the gel with water, freezing, and then freeze drying under avacuum. A typical procedure may be used as follows: polymer is added tosolvent such as THF was added to make a solution about 1% (wt/v) to 15%(wt/v). The solution is stirred until uniform. Polymer solution(prewarmed to 50° C.) is added into a Teflon vial. The vial containingpolymer solution is then rapidly chilled to gel. The gel-time depends ontemperature, solvent, and the polymer concentration. The gel is kept attemperature for at least 120 minutes. The gel is than immersed intodistilled water for solvent exchange for 2 days. Following solventexchange, the gel is removed from water, dried with filter paper, andfrozen at −18° C. The frozen gel is than transferred into afreeze-drying vessel at about −10° C. under vacuum lower than 0.5 mm Hgfor 1 week. The dried scaffolds are then maintained in a desiccator.

The nanofibers comprising the nanofibrillar structure may comprise oneor more bioactive molecules as described above for the improvednanofiber. The bioactive molecules may be incorporated into thenanofiber network during fabrication of the network or may be attachedto a surface of the network via a functional group. In an embodiment,the polymer or polymer system from which the nanofiber is fabricated maycomprise one or more of the bioactive molecules including, but notlimited to, a lipid, growth factor, differentiation factor, fibrousprotein, and adhesive protein. The lipid may be lysophosphatidylcholine,phosphatidylcholine, sphingomyelin, or mixtures thereof. Preferably thegrowth factor is VEGF, bone morphogenic factor β, EGF, PDGF, NGF, FGF,IGF, or TGF. Preferably the differentiation factor is neurotrophin, CSF,or TGF. Preferably the differentiation factor is neurotrophin, CSF, orTGF.

Functional groups may be incorporated onto a surface of the network asdescribed for the improved nanofiber. The functionalized surfaces of thenetwork may be reacted to bind a peptide, polypeptide, lipid,carbohydrate, polysaccharide, nucleotide, nucleic acid, polynucleotide,or other bioactive molecule to the surface of the network. In anembodiment, the functionalized surfaces of the network are reacted tobind one or more bioactive molecules. Preferably one or more of thebioactive molecules is a growth factor, differentiation factor, fibrousprotein, and/or adhesive protein. Preferably the growth factor is VEGF,bone morphogenic factor β, EGF, PDGF, NGF, FGF, IGF, or TGF. Preferablythe differentiation factor is neurotrophin, CSF, or TGF.

ii. Substrate

Structural properties of the nanofibrillar structure such as strengthand flexibility are provided in large part by the substrate on which thenanofiber network is deposited. The substrate may comprise cellulose,glass or plastic. Preferably the plastic is non-cytotoxic. The substratemay be a film or culture container. Preferably the film has a thicknessof not more than about 10 to about 1000 microns.

The substrate may be water soluble or water insoluble. A substrate thatis water soluble is preferably a polyvinyl alcohol film and can be usedwith a polyvinyl alcohol fiber matrix. The substrate may be porous ornon-porous. Porosity of the substrate is determined by cellularpenetration. A cell is able to penetrate a porous substrate but is notable to penetrate a non-porous substrate. Preferably the pores in aporous substrate have a diameter of about 2 μm to about 10 μm. Thesubstrate may be biodegradable and/or biodissolvable. Preferably thesubstrate is biocompatible.

The substrate may comprise one or more bioactive molecules. Thebioactive molecules may be incorporated into the substrate duringfabrication of the substrate or may be attached to a surface of thesubstrate via a functional group. Functional groups may be incorporatedonto a surface of the substrate as described for the improved nanofiber.The functionalized surfaces of the substrate may be reacted to bind apeptide, carbohydrate, polysaccharide, lipid, nucleotide, nucleic acid,polynucleotide, or other bioactive molecule to the surface of thesubstrate.

In an embodiment, the functionalized surfaces of the substrate arereacted to bind one or more bioactive molecules. Preferably one or moreof the bioactive molecules is a growth factor, differentiation factor,fibrous protein, and/or adhesive protein. Preferably the growth factoris VEGF, bone morphogenic factor β, EGF, PDGF, NGF, FGF, IGF, or TGF.Preferably the differentiation factor is neurotrophin, CSF, or TGF.Preferably the differentiation factor is neurotrophin, CSF, or TGF. Thesubstrate may release one or more bioactive molecules. The rate ofrelease is determined by the rate of dissolution and/or degradation ofthe substrate.

iii. Spacer

Structural properties of a nanofibrillar structure, such as strength andflexibility, may be further provided by a spacer. Spacers may alsoprovide sufficient separation between a nanofiber network and asubstrate or sufficient separation between two or more nanofibrillarstructures to permit cells to penetrate and attach to the nanofibers.

In an embodiment, the spacer comprises a first and second surfacewherein the first surface of the spacer contacts a surface of thenanofiber network deposited on a substrate and the second surface of thespacer contacts a surface of the substrate such that the nanofibernetwork and substrate are separated by the diameter or thickness of thespacer. In another embodiment, the spacer comprises a first and secondsurface wherein the first surface of the spacer contacts a surface of afirst nanofibrillar structure and the second surface of the spacercontacts a surface of a second nanofibrillar structure such that the twonanofibrillar structures are separated by the diameter or thickness ofthe spacer.

The spacer may comprise a fine fiber or film. Preferably the film has athickness of not more than about 10 to about 50 microns. The fine fibermay comprise a microfiber. Preferably the microfiber has a diameter ofabout 1 micron to about 10 microns. The microfiber may be unwoven ornet. The microfiber may be fabricated from many polymers includingcellulose, polyamide, polyester, and polytetrafluoroethylene.

The spacer may be water soluble or water insoluble. The spacer may beporous or non-porous. Porosity of the substrate is determined bycellular penetration. A cell is able to penetrate a porous spacer but isnot able to penetrate a non-porous spacer. Preferably the pores in aporous spacer have a diameter of about 2 μm to about 10 μm. The spacermay be biodegradable and/or biodissolvable. Preferably the spacer isbiocompatible.

The spacer may comprise one or more bioactive molecules. The bioactivemolecules may be incorporated into the spacer during fabrication of thespacer or may be attached to a surface of the spacer via a functionalgroup. Functional groups may be incorporated onto a surface of thespacer as described for the improved nanofiber. The functionalizedsurfaces of the spacer may be reacted to bind a peptide, carbohydrate,polysaccharide, lipid, nucleotide, nucleic acid, polynucleotide, orother bioactive molecule to the surface of the spacer.

In an embodiment, the functionalized surfaces of the spacer are reactedto bind one or more bioactive molecules. Preferably one or more of thebioactive molecules is a growth factor, differentiation factor, fibrousprotein, and/or adhesive protein. Preferably the growth factor is VEGF,bone morphogenic factor β, EGF, PDGF, NGF, FGF, IGF, or TGF. Preferablythe differentiation factor is neurotrophin, CSF, or TGF. Preferably thedifferentiation factor is neurotrophin, CSF, or TGF. The spacer mayrelease one or more bioactive molecules. The rate of release isdetermined by the rate of dissolution and/or degradation of the spacer.

iii. Multi-Layered Assembly of Nanofibrillar Structures

A nanofibrillar structure of the invention may be used in a variety ofapplications, including cell culture and tissue culture applications,high throughput applications for drug discovery, and filtrationapplications. In one application, the nanofibrillar structure may beutilized singly or layered to form a multi-layered nanofibrillarassembly for cell or tissue culture. The nanofibrillar structures havemany in vivo and ex vivo uses including wound repair, growth ofartificial skin, veins, arteries, tendons, ligaments, cartilage, heartvalves, organ culture, treatment of burns, and bone grafts.

A diverse array of growth environments for a cell or tissue may beconstructed by engineering specific chemical and physical propertiesinto the nanofiber network, substrate, and/or spacers comprising theindividual nanofibrillar structure and/or sequentially layeringindividual nanofibrillar structures. Physical properties and/orcharacteristics of the individual nanofibrillar structure including, butnot limited to, texture, rugosity, adhesivity, porosity, solidity,elasticity, geometry, interconnectivity, surface to volume ratio, fiberdiameter, fiber solubility/insolubility, hydrophilicity/hydrophobicity,fibril density, and fiber orientation may be varied and/or modified toconstruct nano- and/or micro-environments that promote one or moreselected cellular activities, including growth and/or differentiation.Specific nano- and/or micro-environments may be engineered withinindividual nanofibrillar structures or within a cellular arraycomprising two or more layered nanofibrillar structures.

Specific recognition motifs such as peptides, polypeptides, lipids,carbohydrates, amino acids, nucleotides, nucleic acids, polynucleotides,or polysaccharides including, but not limited to, growth factors,differentiation factors, fibrous proteins, adhesive proteins,glycoproteins, functional groups, adhesive compounds, deadhesivecompounds, and targeting molecules may be engineered into thenanofibrillar network, substrate, and/or spacers of the individualnanofibrillar structures or multi-layered nanofibrillar assembly eitherisotropically or as gradients to promote appropriate cellular activity,including cell growth and/or differentiation. Embodiments involvingamino acids, peptides, polypeptides, and proteins may include any typeof such molecules of any size and complexity as well as combinations ofsuch molecules. Examples include, but are not limited to, structuralproteins, enzymes, and peptide hormones.

Many types of cells may be grown on the nanofibrillar structureincluding, but not limited to, stem cells, committed stem cells,differentiated cells, and tumor cells. Examples of stem cells include,but are not limited to, embryonic stem cells, bone marrow stem cells andumbilical cord stem cells. Other examples of cells used in variousembodiments include, but are not limited to, osteoblasts, myoblasts,neuroblasts, fibroblasts, glioblasts, germ cells, hepatocytes,chondrocytes, keratinocytes, smooth muscle cells, cardiac muscle cells,connective tissue cells, glial cells, epithelial cells, endothelialcells, hormone-secreting cells, cells of the immune system, and neurons.In some embodiments it is unnecessary to pre-select the type of stemcell that is to be used, because many types of stem cells can be inducedto differentiate in an organ specific pattern by engineering thephysical and/or chemical properties of the individual nanofibrillarstructures or multi-layered nanofibrillar assembly by delivering theindividual nanofibrillar structures or multi-layered assembly to a givenorgan. For example, a stem cell may be induced to become a liver cell byengineering the appropriate differentiation and/or growth factors intothe nanofibrillar structure or by implanting a nanofibrillar structurecomprising stem cells within the liver. Cells in the nanofibrillarstructure can serve the purpose of providing cell seeding, producingcertain compounds, or both.

Cells useful in the invention may be cultured in vitro, derived from anatural source, genetically engineered, or produced by any other means.Any natural source of prokaryotic or eukaryotic cells may be used.Embodiments in which nanofibrillar structures are implanted in anorganism can use cells from the recipient, cells from a nonspecificdonor or a donor from a different species, or bacteria or microbialcells. Cells harvested from a source and cultured prior to use areincluded.

Some embodiments use atypical or abnormal cells such as tumor cells. Thephysical and/or chemical properties of the nanofibrillar structure,including growth and differentiation factors, on which such cells aregrown may be engineered to mimic the native in vivo nano- ormicro-environment of the tumor. Tumor cells cultured on nanofibrillarstructures can provide more accurate representations of the native tumorenvironment in the body for the assessment of drug treatments. Growth oftumor cells on nanofibrillar structures of the invention facilitatecharacterization of biochemical pathways and activities of the tumor,including gene expression, receptor expression, and polypeptideproduction, in an in vivo-like environment allowing for the developmentof drugs that specifically target the tumor.

Some embodiments use cells that have been genetically engineered. Theengineering involves programming the cell to express one or more genes,repressing the expression of one or more genes, or both. One example ofgenetically engineered cells useful in the present invention is agenetically engineered cell that makes and secretes one or more desiredbioactive molecules. When nanofibrillar structure comprising geneticallyengineered cells are implanted in an organism, the molecules producedcan produce a local effect or a systemic effect, and can include themolecules identified above as possible substances. Cells can alsoproduce antigenic materials in embodiments in which one of the purposesof the nanofibrillar structure is to produce an immune response. Cellsmay produce substances to aid in the following non-inclusive list ofpurposes: inhibit or stimulate inflammation; facilitate healing; resistimmuno-rejection; provide hormone replacement; replaceneurotransmitters; inhibit or destroy cancer cells; promote cell growth;inhibit or stimulate formation of blood vessels; augment tissue; and tosupplement or replace skin, synovial fluid, tendons, cartilage,ligaments, bone, muscle, organs, dura, blood vessels, bone marrow, andextracellular matrix.

Genetic engineering can involve, for example, adding or removing geneticmaterial to or from a cell, altering existing genetic material, or both.Embodiments in which cells are transfected or otherwise engineered toexpress a gene can use transiently or permanently transfected genes, orboth. Gene sequences may be full or partial length, cloned or naturallyoccurring.

In an embodiment, viable cells are deposited on a nanofibrillarstructure. Nano- and/or micro-environments that promote cellularactivity of a particular cell or tissue may be engineered into thenanofibrillar structure by varying and/or modifying selected physicaland/or chemical properties of the nanofiber network. The physical and/orchemical properties may be engineered into the individual nanofibrillarstructures as described above. The nanofibrillar structure comprisingthe cells is cultured under conditions that promote cellular activity,including growth and/or differentiation.

In another embodiment, two or more nanofibrillar structures are layeredto form a multi-layered nanofibrillar assembly. Nano- and/ormicro-environments that promote cellular activity of a particular cellor tissue may be constructed by layering nanofibrillar structures thathave selected physical and/or chemical properties. The physical and/orchemical properties may be engineered into the individual nanofibrillarstructures as described above. Viable cells are deposited onto themulti-layered nanofibrillar assembly and the assembly is cultured underconditions that promote growth and/or differentiation of the depositedcells.

In another embodiment, multiple cell types are cultured on individualnanofibrillar structures under different culture conditions and thenassembled, manually or mechanically, layer by layer under sterileconditions into a specific multi-layered nanofibrillar assembly. Nano-and/or micro-environments that promote cellular activity of particularcell types may be engineered within an individual nanofibrillarstructure by varying and/or modifying selected physical and/or chemicalproperties of the nanofibrillar structure or within the assembly byselectively layering the individual nanofibrillar structures to obtainthe desired nano- or micro-environment. The physical and/or chemicalcharacteristics may be engineered as described above. The multi-layerednanofibrillar assembly is than cultured under conditions that promotecellular activity, including cell growth and/or differentiation.

In another embodiment, multiple cell types are cultured on individualnanofibrillar structures under different culture conditions. Thephysical and chemical properties of the individual nanofibrillarstructures may be customized for a particular cell type. The substrateand/or spacers of the nanofibrillar structures are biodegradable and/orbiodesolvable allowing for controlled release of bioactive moleculesduring culture. The bioactive molecules are selected to promote adesired cellular activity, including growth and/or differentiation. Theindividual nanofibrillar structures are then assembled, manually ormechanically, layer by layer under sterile conditions into amulti-layered nanofibrillar assembly. The multi-layered assembly may belayered to create nano- and/or micro-environments that promote a desiredcellular activity, including growth and/or differentiation.Biodegradable and/or biodesolvable spacers comprising selected bioactivemolecules may be inserted between the layered nanofibrillar structurescomprising the assembly to fine tune nano- and/or micro-environmentswithin the assembly. The rate of release of the bioactive molecules fromthe spacer may be determined by the rate of biodegradation and/orbiodissolution of the polymer comprising the spacer. The assembledcellular array is than cultured under conditions that promote cellularactivity, including cell proliferation and/or differentiation.

In another embodiment, nanofibrillar structures are individually wrappedand sterilized. After removal from the packaging, the nanofibrillarstructures may be assembled manually or mechanically, layer by layer,within a culture container to form a multi-layered nanofibrillarassembly.

Degradation and potential redistribution within the body of newmaterials for in vivo applications, such as the nanofibrillar structureand multi-layered nanofibrillar assembly of the invention, need to beevaluated. Materials that are implanted into specific sites within thebody can be degraded and then transported to other regions that aredistant from the original site of incorporation. To examine thedegradation properties of the nanofibers within the body, the nanofibernetwork, substrate, and/or spacers comprising the individualnanofibrillar structures the of the multi-layered assembly may belabeled with a fluorescent marker as described above for the improvednanofiber. In an embodiment, the fluorescent marker is a quantum dot.Because quantum dots are both fluorescent and opaque to magneticresonance imaging, both multi-photon fluorescence microscopy (forexamination of isolated tissues) and MRI (for in vivo analysis) may beused to detect and track the distribution of quantum dot containingfragments or decomposition products of the multi-layered nanofibrillarassembly within tissues and determine whether the decomposition productsof the assembly elicit a significant foreign body response whenincorporated into different tissues or sites within the a body.

iv. Additional Uses

In another application, the nanofibrillar structures of the inventionmay be used in high throughput applications for analyzing drug/cellinteractions. High throughput applications are a valuable approach fordiscovery of new pharmaceuticals. High throughput applications utilizemultiwell tissue culture chambers with densities up to about 1536 wellsper plate. Increasing the population of cells per well would serve toincrease the measured signals. In an embodiment, nanofibrillarstructures may be inserted into a well. In another embodiment, a surfaceof the well may function as the substrate allowing the nanofiber networkto be deposited directly onto a surface of the well. The introduction ofsuch nanofibrillar structures into the wells provides additionalsurfaces for cell, ligand, and/or enzyme attachment without affectingthe ability to perform optical measurements.

In another application, the nanofibrillar structure may be used inpurification and/or separation applications. Individual nanofibrillarstructures may be layered to form a chromatography column. Physicaland/or chemical properties including size, charge, hydrophobicity, andaffinity for other molecules may be exploited to separate, for example,a protein from a solution. In an embodiment, individual nanofibrillarstructures may be layered to form an affinity column. Physical and/orchemical properties including, for example, specific functional groups,polypeptides comprising a specific receptor, or immunoglobulins, may beengineered into or attached to the nanofibrillar structures toselectively bind specific polypeptides from a solution. The solution ispassed through the multi-layered nanofibrillar assembly allowing thepolypeptides to bind to the matrix. The bound polypeptides may bereleased from the assembly with a solvent. In another embodiment,individual nanofibrillar structures may be layered to form a filtrationcolumn. Physical properties including, for example, porosity and fibrildensity may be engineered into the multi-layered nanofibrillar assemblyto allow polypeptides to be separated from a solution according to theirsize. In another embodiment, individual nanofibrillar structures may belayered to form an ion-exchange column. Chemical properties including,for example, negatively and/or positively charged functional groups maybe engineered into or attached to the nanofibrillar structures.

In another application, the structures of the invention may be used tofabricate a bioreactor.

C. Cell Growth Media

Another aspect of the invention is a cell growth media. The cell growthmedia may comprise a matrix, mat, network, sheet, or roll. In anembodiment, the media comprises a matrix of nanofibers. The nanofibersmay be fabricated from a polymer or polymer system as described abovefor the improved nanofiber. The cell growth media may be deposited onthe surface of a culture container or into a culture container.

The growth media may be fabricated to desired dimensions for use in aculture container. In an embodiment, the cell growth media comprises amatrix of nanofibers wherein the network has a fiber diameter of about50 nm to about 1000 nm, an average interfiber spacing of at least about2 microns, a matrix solidity of about 30 percent or less, and a top anda bottom with an outerwall wherein the outerwall has a height of about10 microns to about 100 mm and wherein the top and bottom independentlyhave an area of about 5 mm² to about 4×10⁵ mm². The growth media may besized or resized to selected dimensions for use in a culture containerby cutting down to size a sheet, roll, matrix, network, or matcomprising the growth media or cutting a piece with the desireddimensions from a matrix, sheet, roll, network, or mat comprising thegrowth media. In another embodiment, the cell growth media comprises anetwork of nanofibers wherein the dimensions of the network are adaptedfor insertion into a culture container.

The cell growth media may be used in a variety of applications,including cell culture and tissue culture applications. Any celldescribed above may be grown on the cell growth media. In an embodiment,cell growth media is individually wrapped and sterilized. After removalfrom the packaging, the media may be placed within a culture containerto form a surface for cell growth. In another embodiment, cell growthmedia is deposited onto an inside surface of a culture container. Theculture containers comprising the cell growth matrix may be individuallywrapped and sterilized. After removal from the packaging, cells may bedeposited onto the cell growth media within the culture container. Cellsgrown in the cell growth media have many in vivo and ex vivo usesincluding wound repair, growth of artificial skin, veins, arteries,tendons, ligaments, cartilage, heart valves, organ culture, treatment ofburns, and bone grafts.

The physical properties and/or characteristics of the cell growth mediaincluding, but not limited to, texture, rugosity, adhesivity, porosity,elasticity, solidity, geometry, and fibril density may be varied and/ormodified to promote a desired cellular activity, including growth and/ordifferentiation. Specific nano- and/or micro-environments may beengineered within the cell growth media. For example, the porosity andfibril density of the cell growth media may be varied and/or modified toallow a cell to penetrate the cell growth media and grow in a threedimensional environment. The physical properties of the cell growthmedia may be engineered as described above for the improved nanofiberand/or nanofibrillar structure.

Specific recognition motifs such as peptides, lipids, carbohydrates,amino acids, nucleotides, nucleic acids, polynucleotides, orpolysaccharides including but not limited to growth factors,differentiation factors, fibrous proteins, adhesive proteins,glycoproteins, functional groups, adhesive compounds, deadhesivecompounds, and targeting molecules may be engineered into the cellgrowth media either isotropically or as gradients to promote desiredcellular activity, including cell growth and/or differentiation. Thechemical properties of the cell growth media may be engineered asdescribed above for the improved nanofiber and/or nanofibrillarstructure.

In another application, the cell growth media of the invention may beused in high throughput applications for analyzing drug/cellinteractions. High throughput applications utilize multiwell tissueculture chambers with densities up to about 1536 wells per plate.Increasing the population of cells per well would serve to increase themeasured signals. In an embodiment, cell growth media may be insertedinto the wells of the tissue culture chamber used for the analysis. Inanother embodiment, a surface of the well may function as a substratefor the cell growth media allowing a nanofiber network or matrix to bedeposited directly onto a surface of the well. The introduction of suchcell growth into the wells provides additional surfaces for cell,ligand, and/or enzyme attachment without affecting the ability toperform optical measurements.

EXAMPLES

The invention is illustrated by the following Examples, which serve toexemplify the embodiments. Many variations and embodiments, however, canbe made to the disclosed invention. The Examples are not intended tolimit the invention in any way.

Example 1 Electrospinning a Polymer Solution Comprising a Lipid Producesan Enhanced Population of Thin Fibers

To visualize the changes in fiber diameter associated with the additionof a lipid to a polymer solution using an optical microscope, fiberswere electrospun to obtain microfibers. Microfibers were electrospunfrom a solution comprising 15% poly(ε-caprolactone) (w/w) in chloroformsupplemented with (Dow Tone Polymers, Midland, Mich.) 0, 0.25, 0.5, 1.0and 1-% respectively of cholesterol (w/w) (Sigma, St. Louis, Mo.). Thefibers were electrospun using a capillary needle system. An Eppendorfmicropipiette tip (yellow) was fitted to a 5 cc syringe. The polymersolution was poured into the syringe and a positive electrode connectedto a Nanosecond Optical Pulse Radiator Model NR-1 (Optitron, Inc.,Torrance, Calif.) was inserted into the solution. The electrospinningvoltage was 18,000 volts. The fibers were electrospun onto a groundedmetal plate target spinning in a plane perpendicular to the syringe. Thetarget was placed 2 inches from the tip of the micropipetter. The fiberswere collected on overhead transparency placed on the target. The fiberswere viewed on a light microscope (Insight Bilateral Scanning ConfocalFluorescence Microscope (Meridian Instruments, Okemos, Mich.) with a 20×objective and the images were digitally acquired with a CCD camera.

As shown in FIGS. 2A-E, addition of cholesterol to the polymer solutionproduces fibers with progressively smaller diameters. Increasing theamount of cholesterol added to the polymer solution produced fibers withprogressively smaller diameters. Fibers produced from a polymer solutioncomprising no cholesterol had a range of diameters from of 10-50microns. (FIG. 2A). Fibers produced from a polymer solution comprising0.25% cholesterol (w/w) had a range of diameters from 5-30 microns.(FIG. 2B). Fibers produced from a polymer solution comprising 0.5%cholesterol (w/w) had a range of diameters from 2-20 microns. (FIG. 2C).Fibers produced from a polymer solution comprising 1.0% cholesterol(w/w) had a range of diameters from 1-15 microns. (FIG. 1D). Fibersproduced from a polymer solution comprising 10% cholesterol (w/w) had adiameter of 0.8-8 microns. (FIG. 2E).

Example 2 Nanofibers Comprising a Lipid Induce Tight Attachment of Cellsto the Nanofiber

Nanofibers comprising a lipid provide a surface that promotesrecruitment of cells and tight association between the cells andnanofibers. Normal kidney rat (NRK) fibroblasts were cultured onnanofibers electrospun from a solution comprising 10%poly(ε-caprolactone) (w/w) in chloroform supplemented with 0.25%sphingomyelin in Dulbecco Modified Eagle's Medium (DME) at 37° C. in 5%CO₂ and visualized on a light microscope (Insight Bilateral ScanningConfocal Fluorescence Microscope (Meridian Instruments, Okemos, Mich.))with a 20× objective. Images were captured with a CCD camera.

As shown in FIGS. 3A and B, nanofibers comprising 0.25% sphingomyelininduce a rapid recruitment of cells and their attachment to thenanofibers. FIG. 3A shows NRK fibroblasts after two days of culture on atissue culture plate coated with nanofibers comprising 0.25%sphingomyelin. The fibroblasts are tightly attached to the nanofibersand have spread and divided to fill the spaces between the fibersforming a monolayer. FIG. 3B shows NRK fibroblasts after two days ofculture on a tissue culture plate coated with nanofibers not containingsphingomyelin. Few cells are attached to the nanofibers. The guidedmigration and tight attachment properties of the lipo-containingnanofibers suggest the fibers may be useful in vivo or ex vivo forapplications including wound repair, growth of artificial skin, veins,arteries, tendons, ligaments, cartilage, heart valves, organ culture,treatment of burns, and bone grafts

Example 3 Cells Grown on Nanofiber Network have Actin Networks Similarto Cells within Tissue

The actin network of a cell has been utilized as a marker to determinewhich cell culture methods most closely approximate the environmentswithin tissues (Cukierman et al., 2001, Science, 23:1708-1712; Walpitaand Hay, 2002, Nature Rev. Mol. Cell. Biol., 3:137-141). When grown intwo-dimensional tissue culture, fibroblasts assume a highly spread andadhering morphology in which the actin network located within thecytoplasm is organized into arrays of thick stress fibers. In contrast,fibroblasts observed in tissues are spindle-like in shape with actinorganized in a cortical ring (Walpita and Hay, 2002, Nature Rev. Mol.Cell. Biol., 3:137-141).

We compared the actin network of normal rat kidney (NRK) fibroblastsgrown on two-dimensional and three-dimensional surfaces. Fibroblastswere grown on polyamide nanofiber network, glass, and glass coated withpolylysine. The polyamide nanofibers were spun using either a rotatingemitter system or a capillary needle system. Both systems producesubstantially the same fibrous materials. The flow rate was 1.5 mil/minper emitter, a target distance of 8 inches, an emitter voltage of 88 kV,a relative humidity of 45%, and for the rotating emitter an rpm of 35.

Formation of actin networks was monitored by imaging the distribution ofgreen fluorescent protein (GFP)-actin chimera expressed by the NRKfibroblasts. Briefly, NRK fibroblasts transfected with GFP-actin (giftfrom Dr. Sanford Simon, Laboratory of Cellular Biophysics, RockefellerUniversity, New York, N.Y.) were cultured on polyamide nanofibers,glass, or glass coated with poly 1-lysine in Dulbecco Modified Eagle'sMedium (DME) at 37° C. in 5% CO₂ and than examined using an InsightBilateral Scanning Confocal Fluorescence Microscope (MeridianInstruments, Okemos, Mich.).

As shown in FIGS. 4A-C, cells grown on the three-dimensional growthenvironment of the nanofiber network organize cytoskeletal networkssimilar to those observed in vivo. Fibroblasts grown on a nanofibernetwork demonstrated a more spindle like morphology with filipodia anddemonstrated few stress fibers. (FIG. 4A) In contrast, fibroblasts grownon a glass surface were spread with many stress fibers. (FIG. 4B).Fibroblasts grown on a poly 1-lysine coated glass surface were morespread and showed thicker and more pronounced stress fibers (FIG. 4C).

These micrographs also demonstrate that confocal microscopy offers arapid means to monitor differences in the organization of cytoskeletalnetworks in live cells or tissues as a function of various fibermanipulations including physical properties of the fiber networkincluding, but not limited to, texture, rugosity, adhesivity, porosity,elasticity, geometry, and fibril density, and chemical properties of thefiber network including, but not limited to, growth factors,differentiation factors, fibrous proteins, adhesive proteins,glycoproteins, functional groups, adhesive compounds, deadhesivecompounds, and targeting molecules. Comparison of cytoskeletal networksof cells or tissues cultured on a nanofiber network with in vivoobservations allows fine-tuning of physical and/or chemical propertiesof the nanofiber network to more closely mimic the in vivo environmentof a cell or tissue.

Example 4 Incorporation of Functional Groups into Nanofibers

Functional groups such as alcohol, aldehyde, amino, carboxy, andsulphydryl functional groups and photoactivatable functional groups,such as carbene or nitrene may be incorporated onto the surface ofnanofibers. These groups may be used to covalently couple bioactivemolecules including, but not limited to, polypeptides such as growthfactors or differentiation factors, carbohydrates, lipids,polysaccharides, or therapeutic drugs. Functional groups may beincorporated into a nanofiber by adding the functional groups into thepolymer solution. Nanofibers were electrospun from a solution comprising10% poly(ε-caprolactone) (w/w) in chloroform supplemented with (Dow TonePolymers, Midland, Mich.) 2% dodecyl amine (w/w) (Sigma, St. Louis, Mo.)as described for Example 1. To demonstrate the availability ofmodifiable amines at the surface of the nanofibers, the nanofibers werereacted with fluorescein isothiocyanate (1 mg/ml stock solution inwater) (Sigma, St. Louis, Mo.) in 2.0% sodium phosphate buffer, pH 8.5.Incorporation of fluorescence into the fibers was shown utilizing anInsight Bilateral Scanning Confocal Fluorescence Microscope (MeridianInstruments, Okemos, Mich.). As shown in FIG. 5A, a low level offluorescence was observed on the surface of unmodified fibers that is aresult of unreacted fluorescein isothiocyanate adsorbed onto the fibersurface. In contrast, on nanofibers containing incorporated aminogroups, significant fluorescence was observed along the entire fiberfollowing reaction with fluorescein isothiocyanate (FIG. 5B).

This data demonstrates that the modified surfaces of the nanofiberscontain functional groups that are available for coupling with bioactivemolecules allowing for customized incorporation of molecules such asgrowth factors, differentiation factors, fibrous proteins, adhesiveproteins, glycoproteins, functional groups, adhesive compounds,deadhesive compounds, and targeting molecules to create a nano- and/ormicro-environment within the nanofiber network that promotes one or moreselected activities in a cell or tissue, including growth and/ordifferentiation. This data also suggests that nanofibers comprisingdifferent functional groups in specific spatial and geometricarrangements may be induced to self-assemble into a geometricallydefined array of peptide derivatized fibers by introducing peptideshaving the appropriate reactive groups.

Example 5 Labeling Fine Fibers with Quantum Dots

In order to prepare fibers with different chemical and/or physicalproperties and incorporate them into nanofiber blends or specificcellular arrays while still maintaining the ability to identify eachtype of fiber within the blend or cellular array, we studiedincorporating fluorescent labels into the nanofiber. A 1% solution ofquantum dots (gift of Dr. Sanford Simon, Laboratory of CellularBiophysics, Rockefeller University, New York, N.Y.) was added to apolymer solution comprising 12% poly(ε-caprolactone) (w/w) inchloroform. The solution was then electrospun as described for Example 1producing a population of microfibers containing quantum dots. Thefibers were excited at 488 nm and images of the quantum dot distributionwithin the microfibers were collected using an Insight BilateralScanning Confocal Fluorescence Microscope (Meridian Instruments, Okemos,Mich.) (FIG. 6).

Fluorescence of quantum dots is quenched in aqueous environments.Addition of water to the microfibers did not quench the fluorescence ofthe quantum dots, demonstrating that the quantum dots were embeddedwithin the fiber matrix rather than absorbed to the surface of thefiber, an important consideration for their use in aqueous systems.

The above specification, examples and data provide an explanation of theinvention. However, many variations and embodiments can be made to thedisclosed invention. The invention is embodied in the claims hereinafter appended.

1. A method for manufacturing a tissue comprising: layering two or morenanofibrillar structures to form a multi-layered nanofibrillar assemblycomprising an environment for growth of living cells in cell culture,the nanofibrillar structures comprising one or more synthetic polymericnanofibers and a non-cytotoxic substrate, wherein the nanofibrillarstructure is defined by a network of one or more nanofibers, the networkhaving a thickness of a single nanofiber to about 2000 nanometers,wherein the nanofibrillar structure is deposited on a surface of thesubstrate and comprises one or more nanofibers having a diameter ofabout 50 to about 1000 nanometers, an average interfiber spacing ofabout 0.01 microns to about 25 microns, and a solidity of 30 percent to70 percent; depositing viable cells onto the assembly; and culturing thecells deposited onto the assembly under conditions that promote growthand/or differentiation of the deposited cells.
 2. The method of claim 1,wherein the individual nanofibrillar structures are deposited as layers,each layer being separated by a spacer.
 3. The method of claim 2,wherein the spacer comprises a porous film, biodegradable film,microfiber, unwoven, or net.
 4. The method of claim 2, the spacer havinga thickness and a first and second surface wherein the first surface ofthe spacer contacts a surface of a first nanofibrillar structure and asecond surface of the spacer contacts a surface of a secondnanofibrillar structure such that the first and second nanofibrillarstructures are separated by the thickness of the spacer.
 5. The methodof claim 1, wherein the individual nanofibrillar structures comprise oneor more growth factors, differentiation factors, adhesive compounds,deadhesive compounds, and/or targeting compounds.
 6. The method of claim5 wherein the one or more growth factors, differentiation factors,adhesive compounds, deadhesive compounds, and/or targeting compounds ofeach individual nanofibrillar structure are selected to promote thegrowth and/or differentiation of a specific cell type.
 7. The method ofclaim 5, wherein: at least one of the one or more growth factors isvascular endothelial growth factor, bone morphogenic factor β, epidermalgrowth factor, endothelial growth factor, platelet-derived growthfactor, nerve growth factor, fibroblast growth factor, insulin growthfactor, insulin-like growth factor, or transforming growth factor; or atleast one of the one or more differentiation factors is neurotrophin,colony stimulating factor, bone morphogenic factor, or transforminggrowth factor; or at least one of the one or more adhesion molecules arefibronectin or laminin or adhesion peptides derived from thesemacromolecules.
 8. The method of claim 1, wherein the individualnanofibrillar structures present or release the one or more growthfactors or the one or more differentiation factors.
 9. The method ofclaim 8, wherein the rate of release is determined by the rate ofdissolution or degradation of the substrate or nanofibers.
 10. Themethod of claim 1, wherein the one or more nanofibers comprise polyesteror polyamide.
 11. The method of claim 10, wherein the polyester ispoly(epsilon caprolactone), poly(glycolate), or poly(lactate).
 12. Themethod of claim 10, wherein the polyamide is a nylon.
 13. The method ofclaim 1, wherein the one or more nanofibers comprise one or morealdehyde, alcohol, amine, sulphydryl, or photoreactive functionalgroups.
 14. The method of claim 1, wherein the individual nanofibrillarstructures comprise a solidity of 30 percent to 50 percent.
 15. Themethod of claim 1, wherein the substrate is plastic or glass.
 16. Themethod of claim 1, wherein the substrate is a film.
 17. The method ofclaim 16, wherein the film is a polyvinyl alcohol film.
 18. The methodof claim 1, wherein one or more the individual nanofibrillar structuresfurther comprise one or more bioactive molecules.
 19. The method ofclaim 18, wherein at least one of the one or more bioactive molecules isa lipid, carbohydrate, polysaccharide, or polynucleotide molecule. 20.The method of claim 1, wherein at least one of the individualnanofibrillar structures forming the multi-layered nanofibrillarassembly comprises an average interfiber spacing of at least 2 microns.21. The method of claim 1, wherein the network of the one of morenanofibers comprises a thickness of about 100 nanometers to about 1000.22. A method for manufacturing a tissue comprising: culturing aplurality of cells on individual nanofibrillar structures wherein eachindividual nanofibrillar structure contains a specific cell type, thenanofibrillar structures comprising one or more synthetic polymericnanofibers and a non-cytotoxic substrate, wherein the nanofibrillarstructure is defined by a network of one or more nanofibers, the networkhaving a thickness of a single nanofiber to about 2000 nanometers,wherein the nanofibrillar structure is deposited on a surface of thesubstrate and comprises one or more nanofibers having a diameter ofabout 50 to about 1000 nanometers, an average interfiber spacing ofabout 0.01 microns to about 25 microns, and a solidity of 30 percent to70 percent; layering two or more of the individual nanofibrillarstructures to form a multi-layer array of cells; and culturing the arrayunder conditions that promote proliferation and/or differentiation ofthe cells.